This is a renewal application for a research program that focuses on the development and application of deoxyribozymes for phosphorylation and dephosphorylation of proteins. Deoxyribozymes (DNA enzymes) are specific DNA sequences that have particular catalytic activities, just like protein enzymes are catalytic sequences of amino acids. DNA has many conceptual and practical advantages as a catalyst relative to proteins and RNA. At present, much effort has focused on deoxyribozymes for RNA cleavage and ligation, but a broader range of reaction chemistries remains largely unexamined. Expanding DNA catalysis to include protein substrates for the key reactions of phosphorylation and dephosphorylation will increase our fundamental understanding of such catalytic processes and provide practical catalysts for applications involving these important biomolecular substrates. Our previous work has established that deoxyribozymes can have very high rate enhancements for challenging reactions such as DNA phosphodiester hydrolysis. However, most previous efforts by us and others have focused on substrates that are oligonucleotides, for which binding interactions are provided by simple Watson-Crick base pairing.
In Aim 1, we will establish a more general, modular approach in which small-molecule substrates are bound by generalizable aptamer domains, which will then be integrated with catalytic domains to provide functional deoxyribozymes.
Aims 2 and 3 will focus on identification of deoxyribozymes that catalyze protein phosphorylation and dephosphorylation (i.e., kinase and phosphatase activities), respectively.
Aim 4 will apply the new kinase and phosphatase deoxyribozymes in biologically relevant contexts such as cell-surface modification reactions and studies of tau protein, which is implicated in Alzheimer's disease. Finally, Aim 5 will use biochemical methods, solution-state NMR spectroscopy, and X-ray crystallography to characterize the newly identified kinase and phosphatase deoxyribozymes, providing important fundamental information that will assist the rational redesign of selection strategies and catalytic activities.
Catalytic DNA molecules (deoxyribozymes) are an intriguing new form of catalyst that can be used to increase our basic understanding of nucleic acids and to enable practical applications. The proposed research expands the chemical scope of DNA catalysis to include phosphorylation and dephosphorylation of protein substrates, providing fundamental insights as well as practical catalysts for immediate biological applications.
|Brandsen, Benjamin M; Velez, Tania E; Sachdeva, Amit et al. (2014) DNA-catalyzed lysine side chain modification. Angew Chem Int Ed Engl 53:9045-50|
|Chu, Chih-Chi; Wong, On Yi; Silverman, Scott K (2014) A generalizable DNA-catalyzed approach to peptide-nucleic acid conjugation. Chembiochem 15:1905-10|
|Dokukin, Victor; Silverman, Scott K (2014) A modular tyrosine kinase deoxyribozyme with discrete aptamer and catalyst domains. Chem Commun (Camb) 50:9317-20|
|Brandsen, Benjamin M; Hesser, Anthony R; Castner, Marissa A et al. (2013) DNA-catalyzed hydrolysis of esters and aromatic amides. J Am Chem Soc 135:16014-7|
|Parker, Darren J; Xiao, Ying; Aguilar, John M et al. (2013) DNA catalysis of a normally disfavored RNA hydrolysis reaction. J Am Chem Soc 135:8472-5|
|Walsh, Shannon M; Sachdeva, Amit; Silverman, Scott K (2013) DNA catalysts with tyrosine kinase activity. J Am Chem Soc 135:14928-31|
|Chandrasekar, Jagadeeswaran; Silverman, Scott K (2013) Catalytic DNA with phosphatase activity. Proc Natl Acad Sci U S A 110:5315-20|
|Sachdeva, Amit; Chandra, Madhavaiah; Chandrasekar, Jagadeeswaran et al. (2012) Covalent tagging of phosphorylated peptides by phosphate-specific deoxyribozymes. Chembiochem 13:654-7|
|Xiao, Ying; Wehrmann, Rebecca J; Ibrahim, Nora A et al. (2012) Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA. Nucleic Acids Res 40:1778-86|
|Sachdeva, Amit; Silverman, Scott K (2012) DNA-catalyzed reactivity of a phosphoramidate functional group and formation of an unusual pyrophosphoramidate linkage. Org Biomol Chem 10:122-5|
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